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N V Tsupkina

Publications and source records attributed to N V Tsupkina.

At least 19 recordsLinked to original sources
PubMed2005

[Participation of transfused bone marrow cells in reparative osteohistogenesis].

The participation of skeletal tissue cell precursors in the repairing regeneration of bone tissue was studied. Bone marrow was taken from donor animals--mice of C57Bl/6-TgN(ACTbGFP) 1 Osb line (The Jackson Laboratory Bar Harbor ME USA line). Nucleated cell fraction was isolated by centrifugation on a density percoll gradient. Recipient mice C57Bl/6 line were irradiated by 7.0-7.5 Gr dose. Intravenous infusion of donor cells and osteoclasts of tibia was done after irradiation of recipient mice. Histological preparations of bone regenerate tissues were studied on 15, 30, and 60 days by confocal microscopy. Donor cells were found as skeletal tissue precursors into periost, endost, bone marrow, and as differentiated cells of newborn tissue of regenerate--osteoblasts, osteocytes, chondrocytes. The data obtained indicate that part of donor bone marrow cells are able to progressive differentiation under recipient bone fractures.

Animals↗
PubMed2005

[EGF-dependent signaling pathways are activated under heat stress in A431 carcinoma cells].

EGF receptor transactivation and activation of EGF-dependent signaling pathways under heat shock conditions were studied. Heating A431 cells at 42 degrees C induced both EGF receptor tyrosine phosphorylation and appearance of phosphorylated forms of key components of its downstream signaling pathways - phospholipase Cgamma1 (PLCgamma1), transcription factor STAT3, and EPK1/2. It is suggested that EGF receptor is transactivated under heat shock in A431 cells. Pretreatment of heat-shocked cells with a specific inhibitor of EGF receptor tyrosine kinase tyrphostin AG1478 does not prevent EGF receptor and EPK 1/2 tyrosine phosphorylation. In contrast, tyrphostin AG1478 abrogates tyrosine phosphorylation of PLCgamma1 and STAT3. This suggested that the intrinsic EGF receptor tyrosine kinase is not involved in EGF receptor transactivation, but is sufficient for PLCgamma1 and STAT3 activation in stress conditions. The effect of a conditioned medium of heated cells was investigated to check whether autocrine mechanism is involved in EGF receptor transactivation. The conditioned medium of heated cells induced both tyrosine phosphorylation of EFG receptor and ERK 1/2. Simultaneously, neither PLCgamma1, not STAT3 phosphorylation were detected. Here, for the first time, we demonstrated the involvement of autocrine mechanism in EGF receptor transactivation under heat stress in A431 carcinoma cells, but additional intracellular events are essential for activation of EGF receptor downstream signaling pathways.

Carcinoma↗
PubMed2004

[Development and morphofunctional characterization of the osteoblastic phenotype in cell culture in vitro].

The objective of this research was to study osteogenic properties of cultured rabbit bone marrow stromal cells, newborn rat cranium bone cells and rat osteocarcoma ROS 17-2/8 cells. For this purpose cytochemical reaction for alkaline phosphatase was performed by the Lowry method, mineral deposition was assessed by staining of the cultures after von Kossa. Cranium bone cells were shown to synthesize alkaline phosphatase (34 +/- 7 nmol/min/10(6) cells), the matrix mineralization being found. Bone marrow stromal cells displayed a lower activity alkaline phosphatase level than did cranium bone cells (4 +/- 0.6 nmol/min/10(6) cells). However, cell cultivation in the presence of dexamethasone in the medium (10(-8) M) induced a higher activity of alkaline phosphatase (9 +/- 1 nmol/min/10(6) cells), mineralization of the extracellular matrix being the case. The highest level of alkaline phosphatase activity was found for ROS 17-2/8 cells (60 +/- 12 nmol/min/10(6) cells) but no matrix mineralization was determined. According to these data, matrix calcification and formation of bone-like nodules are the most important properties of osteoblastic differentiation in vitro.

Alkaline Phosphatase↗
PubMed2004

[Lamellar bone culture in vitro].

The objective of this study was to analyze morpho-functional characteristics of the cells in definitive osseous tissue cultured in vitro. Cortical plates, isolated from the fragments of iliac bones of adult rabbits, were cultured in vitro in a nutrient medium. The culture obtained was fixed after 3, 10 and 20 days and processed for scanning electron microscopy. To identify the osteogenic cells, alkaline phosphatase activity was demonstrated. It was established that the processes of cell proliferation and migration from the cultured bone pieces were most active at 10 days after the explanation. The growing cells formed a growth zone around the bone fragment that consisted of three layers, differing in the degree of cellular proliferation and differentiation. After 20 days in culture the cells expressing alkaline phosphatase were shown to form groups. The cells of osteon canals in culture were found to possess potential for proliferation and osteogenic differentiation.

Alkaline Phosphatase↗
PubMed2003

[Effect of EGF on ubiquitination and proteasome-dependent degradation of phospholipase C gamma1 in A431 cells].

Phospholipase C gamma 1 (PLC gamma 1), an enzyme participating in phosphoinositide turnover, is one of the key elements in cell signaling. Here it is shown that treatment of A431 carcinoma cells with proteasome inhibitors Mg132 and lactacystin results in increasing the PLC gamma 1 intracellular level. Simultaneously, several additional bands with lower electrophoretic mobilities were detected on immunoblots, using anti-PLC gamma 1 antibodies. PLC gamma 1 ubiquitinilation was shown using immunoprecepitation. In control A 431 cells, PLC gamma 1 is ubiquitinilated, but the addition of EGF greatly induces the ubiquitinilation of the protein. Association of PLC gamma 1 with ubiquitin-ligase c-Cb1 was shown. Dynamics of ubiquitinilation under EGF treatment is in a close agreement with that of association of PLC gamma 1 and c-Cb1. It is concluded that PLC gamma 1 is ubiquitinilated and degraded by proteasomes. PLC gamma 1 ubiquitinilation is an EGF-dependent process.

Acetylcysteine↗
PubMed2003

[Effect of EGF on the intracellular distribution of heat-shock proteins in A431 cells].

The intracellular distribution of hsp70 and hdj1 was studied using immunofluorescent method. In nonstimulated cells hsp70 and hdj1 were observed in the cytoplasm of A431 cells. When 100 ng/ml EGF was added for 15 min, both hsp70 and hdj1 were accumulated in the nuclei. Later on (up to 1 h) hsp70 was exported from the nuclei to be observed mainly in the cytoplasm, whereas hdj1 remained in the nuclei. In cells exposed to tyrphostin AG1478, this inhibitor of tyrosine kinase activity of EGF receptor prevented EGF-dependent accumulation of hsp70 and hdj1 in the nuclei. U73122, an inhibitor of phospholipase C activity, induced tyrosine phosphorylation of EGF receptor without EGF stimulation. In cells treated with U73122, both hsp70 and hdj1 were detected in the nuclei of non-stimulated cells. It is concluded that the intracellular distribution of heat shock proteins in A431 cells depends on tyrosine kinase activity of EGF receptor. Here we report for the first time the influence of EGF on the intracellular redistribution of heat shock proteins.

Antibodies, Monoclonal↗
PubMed2002

[EGF receptor degrades via the proteasome-dependent pathway in A-431 cells].

It is known that a lot of cell receptors degrade by ubiquitine-proteasome pathway. Here we show that degradation of the epidermal growth factor (EGF) receptor is proteasome-dependent. Treatment of A-431 cells with lactacystine, an inhibitor of proteolytic activities of 26S proteasomes, induces accumulation of the receptor in cells. Incubation of cell lysates with isolated 26S proteasomes leads to diminishing EGF receptor in these cells. Active (tyrosine phosphorylated) EGF receptor is a target of proteolysis by proteasomes.

Blotting, Western↗
PubMed2002

[Cell interaction with extracellular proteins during two-phase polymer system formation].

A modified method of investigation of surface properties of cells and proteins with the help of a two-phase polymer system dextran-500/polyethylenglycol-6000 was used. This method is based on changing the kinetics of two-phase system partitioning into phase on adding cells or macromolecules. These changes were registered by measuring the top phase optical density during the system partitioning at 500 nm. Cell lines L (NCTC clone 929), LS and A431, human keratinocytes and platelets, collagen I, laminin-1, laminin-2/4, and fibronectin were studied. The interaction between collagen and all cell types with the formation of complexes takes place during co-partitioning of cells and proteins in the two-phase system. Laminins differ in surface properties and in interaction with cells. Laminin-1 makes preferable complexes with cells of monolayer subline L (NCTC clone 929), but not with cells of suspension subline LS. No interaction of laminin-2/4 with L cells was detected, but, in contrast to laminin-1, this protein has the affinity to A431 cells. No interaction of L cells with fibronectin were detected.

Animals↗
PubMed2001

[Effect of EGF on nuclear-cytoplasmic distribution of proteasomes in A-431 cells].

The intracellular distribution of proteasomes was studied using immunofluorescent method. In nonstimulated cells proteasomes were observed both in the cytoplasm and nuclei of A-431 cells. When 100 ng/ml EGF was added for 15 min, proteasomes were located mainly in the nuclei. Later (up to 1 h) proteasomes released from the nuclei and were observed mainly in the cytoplasm. Tyrphostin AG1478, an inhibitor of tyrosine kinase, and U73122, an inhibitor of phospholipase C, prevent, proteasome export from the nuclei after EGF treatment. In contrast, a proteasome inhibitor--lactacystin has no effect on this process. The EGF-dependent tyrosine phosphorylation of EGF receptor is blocked by tyrhostin AG1478 and U733122. Lactacystin did not alter the induction of EGF receptor tyrosine phosphorylation, triggered by EGF. It is concluded that intracellular distribution of proteasomes depends on tyrosine activity of EGF receptor.

Animals↗
PubMed2001

[Phospholipase C negatively regulates tyrosine phosphorylation of EGF receptor in A-431 cells].

It is known that EGF induces tyrosine phosphorylation and internalization of the EGF receptor in A-431 cells. U73122, an inhibitor of phospholipase C, induces tyrosine phosphorylation of the EGF receptor and its association with phospholipase C still in nonstimulated cells. In U73122 treated cells EGF exerted no effect on these processes. Receptor-mediated endocytosis was not observed in A-431 cells treated with U73122. The reorganization of actin cytoskeleton was detected in U73122 cells.

ErbB Receptors↗
PubMed1999

[Intracellular distribution of proteasomes in A-431 cells].

The intracellular proteasome distribution in A-431 cells was shown using methods of cell fractionation and immunofluorescence. In growing cells the distribution of proteasomes was EGF-dependent. In unstimulated cells and within 30 min of EGF treatment, proteasomes were localized in the cytoplasm and nuclei, but not on the plasma membrane. After 30 min of EGF treatment they were observed on the plasma membrane as well. In A-431 cells cultivated for 24 h in the medium with a lowered serum concentration, proteasomes were detected on the plasma membrane already in unstimulated cells. It is suggested that dephosphorylation of the EGF receptor and signalling proteins in unstimulated cells may depend on the proteolytic activity of proteasomes.

Cell Nucleus↗
PubMed1998

[Intact microtubule network is necessary for the EGF-induced transport of transcription factor STAT1 in the nucleus of A-431 cells].

The mechanism by which transcription factor STAT1 is translocated from the cytoplasm to the cell nucleus is not clear. We put forward a hypothesis suggesting an important role of the cytoskeleton in signal transduction. The results of the present work show that the treatment of cells with nocodazole, a microtubule-disrupting drug, inhibits completely STAT1 import to the nucleus. However, the treatment of cells with cytochalasin B, which is known to depolymerize microfilaments, exerted no detectable effect on the transport of STAT1. The sensitivity to nocodazole treatment suggests that STAT1 may utilize a transport pathway that involves the tubulin cytoskeleton. These data throw light on some mechanism of a rapid and effective nonvesicular transport of STAT1.

Biological Transport↗
PubMed1998

[Association of phosphoinositide-specific phospholipase Cgamma1 with elements of cytoskeleton in A-431 cells].

It is shown that phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1), a substrate of growth factor receptors, is associated with cytoskeleton in A-431 cells. PLC gamma 1 is co-localized only with the cortical actin but not with actin stress fibers. Since EGF receptor is also co-localized with the cortical actin it is concluded that PLC gamma 1 co-localized with actin is mediated by the EGF receptor. After the treatment with cytochalasin B PLC gamma 1 is co-localized with actin aggregates and cytoskeleton elements other than actin. Using double immunofluorescence PLC gamma 1 is shown to be associated with cytokeratin intermediate filaments. The cross-talking of different cytoskeleton elements and their participation in cell signaling is discussed.

Actins↗
PubMed1998

[Participation of phosphoinositide-specific phospholipase Cgamma1 and STAT1 protein in the formation of latent complexes of signal proteins].

It is known that the growth factor activates appropriate membrane receptors which become starting points of cascades of protein-protein interactions leading to cellular response. Recent data suggest that different signalling pathways may cross-talk during the cellular response. Here we show that phosphoinositide-specific phospholipase C gamma 1, one of the key elements in phosphoinositide pathway of signal transduction, is physically associated with members of the STAT pathway. The precipitation of phospholipase C gamma 1, using polyclonal antibody in A-431 cells, leads to co-immunoprecipitation of STAT1 alpha and STAT1 beta, as well as STAT3. The formation of such complexes was observed in both unstimulated and EGF stimulated cells. The participation of SH3-domains in the formation of such complexes is discussed.

Cell Line, Transformed↗
PubMed1997

[The characteristics of protein p66--a substrate of the epidermal growth factor receptor].

The p66-kDa protein--a substrate of EGF receptor, is characterized. It is shown that the molecule of p66 is recognized by antibodies to phospholipase C gamma 1, includes conservative SH2--SH3 domains and lacks N-terminal part of PLC gamma 1. Protein p66 is associated to the EGF-R molecule on 992 tyrosine. Protein p66 binds to both membrane and internalized EGF-Rs and is redistributed with the receptors during receptor-mediated endocytosis. It is supposed that p66 may play a regulatory role in signal transduction.

3T3 Cells↗
PubMed1997

[The effect of nocodazole on the redistribution of phosphoinositide-specific phospholipase C gamma 1 during mitogenic signal transduction in A-431 cells].

Phosphoinositide-specific phospholipase C gamma 1 is associated with EGF receptor in A-431 cells after EGF treatment. It is shown that phospholipase C gamma 1 is co-localized with internalized receptors as well as with membrane ones during receptor-mediated endocytosis. Nocodazole is known as an inhibitor of microtubule assembly, thus leading to a complete disappearance of microtubule network. Nocodazole had no effect on phospholipase C gamma 1--EGF receptor association and co-localization, but the intracellular distribution of both the proteins differed dramatically. Phospholipase C gamma 1 and EGF receptor were localized in endosomes in the periphery of the cell. Besides, pretreatment of A-431 cells with nocodazole resulted in decreasing tyrosine phosphorylation of some proteins. These data suggest that the internalized receptor may serve as an additional starting point for triggering cell signalling.

Antineoplastic Agents↗
PubMed1994

A 66-kDa protein associated with epidermal growth factor receptor is a proteolytic fragment of phosphoinositide-specific phospholipase C.

It is shown that in the A431 cells, EGFR is co-immunoprecipitated with a group of proteins recognized by antibodies to phospholipase C gamma. These are 145- and 47-kDa proteins corresponding to phospholipase C gamma and Nck, respectively, and an unidentified 66-kDa protein. The association of phosphoinositide-specific phospholipase C gamma and 66-kDa protein to EGFR was observed in the A431 cells with or without the EGF treatment. Trypsin peptide maps of these two proteins are similar so it is assumed that the 66-kDa protein is related to phospholipase C gamma.

Antibodies, Monoclonal↗